Patent Application
20220064224
This invention relates to methods and compositions for the control of microorganisms associated with infections in canines and uses thereof.
Domestic dogs and cats, along with wild canine and feline species, can suffer from severe infections caused by viruses of the Parvoviridae family
Amongst infectious diseases, canine parvovirus is one of the leading causes of morbidity and mortality in dogs. Infection results in fever, vomiting, dehydration, and diarrhea. Untreated, most dogs will die from the infection. Current therapies rely on high-cost supportive therapies, such as blood transfusions, IV feeding, and 24-hour care at veterinary hospitals. Canine parvovirus infections are very costly to dog owners, both financially and emotionally. Furthermore, these infections result in lost productivity when individuals stay home from work to care for their dogs. These infections can result in up to 90% mortality in puppies and 10% mortality in adult dogs (Nandi & Kumar, 2010).
The situation is similar in domestic cats, particularly kittens. Feline parvovirus (also known as feline panleukopenia virus) infections results in symptoms that include anemia, fever, vomiting, diarrhea, and decreased white blood cell counts (panleukopenia). Current treatments are high-cost supportive therapies. These infections can result in up to 90% mortality in the acute form of the disease (Stuetzer & Hartmann, 2014).
There is a need for the development of pathogen-specific molecules that inhibit these infections or the association of these pathogens with their hosts.
With reference to the definitions set out below, described herein are polypeptides comprising heavy chain variable region fragments (VHHs) whose intended use includes but is not limited to the following applications in animal health or an unrelated field: diagnostics, in vitro assays, feed, therapeutics, substrate identification, nutritional supplementation, bioscientific and medical research, and companion diagnostics. Also described herein are polypeptides comprising VHHs that bind and decrease the virulence of disease-causing agents in canines or felines. Further to these descriptions, set out below are the uses of polypeptides that comprise VHHs in methods of reducing transmission and severity of disease in host animals, including their use as an ingredient in a product. Further described are the means to produce, characterise, refine and modify VHHs for this purpose.
One aspect of the disclosure includes polypeptide capable of preventing red blood cell hemagglutination by canine parvovirus type 2C capsid at a concentration lower than 100 nM. Another aspect of the disclosure includes a polypeptide capable of preventing red blood cell hemagglutination by canine parvovirus type 2C capsid at a concentration lower than 1 μM. Another aspect of the disclosure includes a polypeptide capable of reducing invasion of MDCK cells by canine parvovirus by >50% at 5 μM. Another aspect of the disclosure includes a polypeptide comprising at least one variable region fragment of a heavy chain antibody (VHH), wherein the at least one VHH specifically binds a parvovirus. In some embodiments, such polypeptide comprises a plurality of VHHs. In some embodiments, the polypeptide comprises at least three VHHs. In some embodiments, any one of the plurality of VHHs is identical to another VHH of the plurality of VHHs. Alternatively and/or additionally, the plurality of VHHs are covalently coupled to one another by a linker, the linker comprising one or more amino acids.
In some embodiments, the variable region fragment of the heavy chain antibody comprises an amino acid sequence at least 80%, 90%, 95%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1 to 18 or 77 to 79. Alternatively and/or additionally, the variable region fragment of the heavy chain antibody comprises a complementarity determining region 1 (CDR1) as set forth in any one of SEQ ID NOs: 19 to 36 or 80 to 82, a complementarity determining region 2 (CDR2) as set forth in any one of SEQ ID NOs: 37 to 54 or 83 to 85, and a complementarity determining region 3 (CDR3) as set forth in any one of SEQ ID NOs: 55 to 72 or 86 to 88.
Another aspect of the disclosure includes a polypeptide complex that comprises a first component polypeptide and a second component polypeptide, wherein the first component polypeptide and the second component polypeptide are not covalently linked together and are coupled together by a protein-protein interaction, a small molecule-protein interaction, or a small molecule-small molecule interaction, wherein each of the first and the second component polypeptides comprise a VHH which specifically binds a pathogen.
In some embodiments, with respect to the polypeptide or the polypeptide complex as described herein, the pathogen is a parvovirus. In some embodiments, respect to the polypeptide or the polypeptide complex as described herein, the parvovirus is from the Parvovirinae subfamily. Alternatively and/or additionally, the parvovirus is from the Densovirinae subfamily. Alternatively and/or additionally, the parvovirus is from the Protoparvovirus genus. Alternatively and/or additionally, the parvovirus comprises a canine parvovirus. Alternatively and/or additionally, the canine parvovirus comprises a canine parvovirus type 2. Alternatively and/or additionally, the canine parvovirus is canine parvovirus type 2, canine parvovirus type 2a, canine parvovirus type 2b, or canine parvovirus type 2c.
In some embodiments, the VHH specifically binds a canine parvovirus virulence factor. Alternatively and/or additionally, the VHH specifically binds an antigen or polypeptide at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ IDs NO: 73 or 75 or 76. Alternatively and/or additionally, the canine parvovirus virulence factor is an infectious canine parvovirus virus particle, a canine parvovirus virus-like particle, or a canine parvovirus capsid protein.
In some embodiments, the parvovirus comprises a feline parvovirus. Alternatively and/or additionally, the parvovirus is feline panleukopenia virus. Alternatively and/or additionally, the VHH specifically binds a feline parvovirus virulence factor. Alternatively and/or additionally, the VHH specifically binds an antigen or polypeptide having a sequence at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 74. Alternatively and/or additionally, the feline parvovirus virulence factor is an infectious feline parvovirus virus particle, a feline parvovirus virus-like particle, or a feline parvovirus capsid protein.
In some embodiments, the parvovirus comprises a mink parvovirus. Alternatively and/or additionally, the parvovirus is mink enteritis virus. Alternatively and/or additionally, the VHH specifically binds a mink parvovirus virulence factor. Optionally, the mink parvovirus virulence factor is an infectious mink parvovirus virus particle, a mink parvovirus virus-like particle, or a mink parvovirus capsid protein Where the parvovirus comprises a mink parvovirus, it is contemplated that the VHH specifically binds an antigen or polypeptide having a sequence at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% identical to SEQ ID NO: 89.
In some embodiments, the parvovirus comprises a swine parvovirus. Alternatively and/or additionally, the parvovirus comprises a mouse parvovirus.
Another aspect of the disclosure includes a nucleic acid encoding the polypeptide or a polypeptide complex described herein. Another aspect of the disclosure includes a plurality of nucleic acids encoding the polypeptide complex described herein. Another aspect of the disclosure includes a vector comprising the nucleic acid encoding the polypeptide or a polypeptide complex or a plurality of nucleic acids encoding the polypeptide complex in any production system.
Another aspect of the disclosure includes a cell comprising the nucleic acid or the plurality of nucleic acids as described herein. In some embodiments, the cell is a yeast cell. Optionally, the yeast is of the genus Pichia. Alternatively and/or additionally, the yeast is of the genus Saccharomyces. In some embodiments, the cell is a bacterial cell. Optionally, the bacteria is of the genus Escherichia. Alternatively and/or additionally, the bacteria is a probiotic bacterium. Alternatively and/or additionally, the probiotic bacteria is selected from the group consisting of the genus Bacillus, the genus Lactobacillus, the genus Bifidobacterium.
In some embodiments, the polypeptide or the polypeptide complex described herein is synthesized in any de novo protein synthesis system. In some embodiments, the polypeptide or the polypeptide complex described herein further comprises meat, a meat by-product, bone meal, fish, fish meal, egg, egg by-product, a vitamin, vegetables, plant matter, plant extracts, an amino acid, a dye, an antibiotic, an antiviral, a hormone, an antimicrobial peptide, a steroid, a prebiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1,3-glucan, vegetable extracts, peptone, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin, gelatin, an additive spray, a toxin binder, a short chain fatty acid, a medium chain fatty acid, an omega-3 fatty acid, yeast, a yeast extract, a plant extract, sugar, a digestive enzyme, a digestive compound, an essential mineral, carnitine, glucosamine, an essential salt, fibre, a preservative, a stabilizer, a natural flavour, an artificial flavour, or water.
Another aspect of the disclosure includes a method of producing the polypeptide or the polypeptide complex described herein, comprising (a) incubating a cell of any one of claims 45 to 52 in a medium suitable for secretion of the polypeptide from the cell; and (b) purifying the polypeptide from the medium.
Another aspect of the disclosure includes the polypeptide or the polypeptide complex described herein for use in reducing or preventing a canine-associated viral infection in a canine, or another animal species. Another aspect of the disclosure includes use of the polypeptide or the polypeptide complex described herein for reducing transmission or preventing transmission of a canine-associated virus from a canine species to another canine or another animal species. In some embodiments, the canine species comprises a domestic dog, wolf, coyote, fox, jackal, or dingo. Alternatively and/or additionally, the another animal species comprises a feline, mink, skunk or raccoon.
Another aspect of the disclosure includes the polypeptide or the polypeptide complex described herein for use in reducing or preventing a feline-associated viral infection in a feline, or another animal species. Another aspect of the disclosure includes use of the polypeptide or the polypeptide complex described herein for reducing transmission or preventing transmission of a feline-associated virus from a feline species to another feline or another animal species. In some embodiments, the feline species comprises a domestic cat, wild cat, leopard, tiger, jaguar, lion, serval, caracal, ocelot, margay, kodkod, oncilla, bobcat, lynx, cheetah, cougar, or jaguarundi. Alternatively and/or additionally, the another animal species comprises a canine, mink, skunk or raccoon.
Another aspect of the disclosure includes the polypeptide or the polypeptide complex described herein for use in reducing or preventing a mink-associated viral infection in a mink, or another animal species. Another aspect of the disclosure includes use of the polypeptide or the polypeptide complex described herein for reducing transmission or preventing transmission of a mink-associated virus from a mink species to another mink or another animal species.
In uses of above, in some embodiments, it is contemplated that the polypeptide is adapted for introduction to the alimentary canal orally or rectally, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the animal dwells (including air based media), provided by injection, provided intravenously, provided via the respiratory system, provided via diffusion, provided via absorption by the endothelium or epithelium, or provided via a secondary organism selected from the group consisting of a yeast, bacterium, algae, bacteriophages, plants and insects to a host.
In describing the present invention, the following terminology is used in accordance with the definitions below.
In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.
Host
As referred to herein, “host”, “host organism”, “recipient animal”, “host animal” and variations thereof refer to the intended recipient of the product when the product constitutes a feed or a therapeutic. In certain embodiments, the host is a vertebrate. In certain embodiments, the host is from the order Carnivora. In certain embodiments, the host is from the family Canidae. In certain embodiments, the host is a domestic dog, wolf, coyote, fox, jackal, or dingo. In certain embodiments, the host is a domestic dog. In certain embodiments, the host is from the family Felidae. In certain embodiments, the host is a domestic cat, a wild cat, leopard, tiger, jaguar, lion, serval, caracal, ocelot, margay, kodkod, oncilla, bobcat, lynx, cheetah, cougar, or jaguarundi. In certain embodiments, the host is a domestic cat. In certain embodiments, the vertebrate host is a non-canine and non-feline species. In certain embodiments, the non-canine and non-feline species is a mink, skunk or raccoon. In certain embodiments, the vertebrate host is a human, swine species, poultry species, ovine species, bovine species, horse, or mouse. In certain embodiments, the host is an invertebrate. In certain embodiments, the invertebrate is a shrimp species.
Pathogens
As referred to herein, “pathogen”, “pathogenic”, and variations thereof refer to virulent microorganisms, that can be associated with host organisms, that give rise to a symptom or set of symptoms in that organism that are not present in uninfected host organisms, including the reduction in ability to survive, thrive, or reproduce. Without limitation, pathogens encompass parasites, bacteria, viruses, prions, protists, fungi and algae. In certain embodiments, the pathogen is a virus belonging to the family Parvoviridae (
“Virulence”, “virulent” and variations thereof refer to a pathogen's ability to cause symptoms in a host organism. “Virulence factor” refers to nucleic acids, plasmids, genomic islands, genes, peptides, proteins, toxins, lipids, macromolecular machineries or complexes thereof that have a demonstrated or putative role in infection.
“Disease-causing agent” refers to a microorganism, pathogen or virulence factor with a demonstrated or putative role in infection.
Antibodies
A schematic of camelid heavy chain only antibodies and their relationship to VHH domains and complementarity determining regions (CDRs) is shown in
As referred to herein “VHH” refers to an antibody or antibody fragment comprising a single heavy chain variable region which may be derived from natural or synthetic sources. NBXs referred to herein are an example of a VHH. In a certain aspect a VHH may lack a portion of a heavy chain constant region (CH2 or CH3), or an entire heavy chain constant region.
As referred to herein “heavy chain antibody” refers to an antibody that comprises two heavy chains and lacks the two light chains normally found in a conventional antibody. The heavy chain antibody may originate from a species of the Camelidae family or Chondrichthyes class. Heavy chain antibodies retain specific binding to an antigen in the absence of any light chain.
As referred to herein “specific binding”, “specifically binds” or variations thereof refer to binding that occurs between an antibody and its target molecule that is mediated by at least one complementarity determining region (CDR) of the antibody's variable region. Binding that is between the constant region and another molecule, such as Protein A or G, for example, does not constitute specific binding.
As referred to herein “antibody fragment” refers to any portion of a conventional or heavy chain antibody that retains a capacity to specifically bind a target antigen and may include a single chain antibody, a variable region fragment of a heavy chain antibody, a nanobody, a polypeptide or an immunoglobulin new antigen receptor (IgNAR).
As referred to herein an “antibody originates from a species” when any of the CDR regions of the antibody were raised in an animal of said species. Antibodies that are raised in a certain species and then optimized by an in vitro method (e.g., phage display) are considered to have originated from that species.
As referred to herein “conventional antibody” refers to any full-sized immunoglobulin that comprises two heavy chain molecules and two light chain molecules joined together by a disulfide bond. In certain embodiments, the antibodies, compositions, feeds, products, and methods described herein do not utilize conventional antibodies.
Production System
As referred to herein, “production system” and variations thereof refer to any system that can be used to produce any physical embodiment of the invention or modified forms of the invention. Without limitation, this includes but is not limited to biological production by any of the following: bacteria, yeast, algae, arthropods, arthropod cells, plants, mammalian cells. Without limitation, biological production can give rise to antibodies that can be intracellular, periplasmic, membrane-associated, secreted, or phage-associated. Without limitation, “production system” and variations thereof also include, without limitation, any synthetic production system. This includes, without limitation, de novo protein synthesis, protein synthesis in the presence of cell extracts, protein synthesis in the presence of purified enzymes, and any other alternative protein synthesis system.
Product
As referred to herein, “product” refers to any physical embodiment of the invention or modified forms of the invention, wherein the binding of the VHH to any molecule, including itself, defines its use. Without limitation, this includes a feed, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic, a drug, a diagnostic tool, a component or entirety of an in vitro assay, a component or the entirety of a diagnostic assay (including companion diagnostic assays).
Feed Product
As referred to herein, “feed product” refers to any physical embodiment of the invention or modified forms of the invention, wherein the binding of the VHH to any molecule, including itself, defines its intended use as a product that is taken up by a host organism. Without limitation, this includes a feed, a pellet, a feed additive, a nutritional supplement, a premix, a medicine, a therapeutic or a drug.
Descriptions of the invention provided are to be interpreted in conjunction with the definitions and caveats provided herein.
Domestic dogs and cats are two of the most popular household animals in the world. Despite the availability of veterinary care and canine vaccines, infectious diseases are still a common problem in dogs and cats. This is particularly true for dogs that spend significant time in kennels, shelter, and dog parks; as well as in puppies from breeding facilities. Significant pathogens affecting dogs include viruses, such as canine parvovirus, rabies, canine distemper virus, canine adenovirus, canine coronavirus, and canine parainfluenza virus, bacteria, such as members of the Bordetella, Borrelia, Brucella, Clostridium, and Leptospira genera, as well as numerous of fungi, protozoa, and parasites. Significant pathogens affecting cats include viruses, such as feline panleukopenia virus, feline immunodeficiency virus, feline leukemia virus, feline herpesvirus 1 (FHV-1), feline calcivirus, and rabies, as well as many bacteria, fungi, and parasites.
Canine parvovirus infections of dogs occur worldwide (Decaro & Buonavoglia, 2012). These infections are acquired by the fecal-oral route and cause diarrhea, vomiting, dehydration, and fever and are often fatal if untreated (Miranda & Thompson, 2016). Supportive therapy consisting of IV treatments, blood transfusions, and around the clock care (Venn et al, 2017) can be a significant financial and emotional burden to dog owners. Similarly, feline parvovirus infections of cats occur around the world. Virus particles can last in the environment for several months, are acquired by cats via the fecal-oral route, cause such symptoms as diarrhea, vomiting, fever, and immunosuppression, have high mortality rates in untreated cats, and are best treated through supportive therapy (Truyen et al, 2009)
To initiate an infection the viral capsid protein interacts with transferrin receptors on the surface of host cell prior to viral invasion (Hafenstein et al, 2007). As such peptides capable of binding the capsid protein at epitopes that are important for host cell binding should be sufficient to neutralize parvovirus infections (Yuan & Parrish, 2000).
Earlier efforts in the field of this invention rely on the host organism to generate protection against disease-causing agents. Although live attenuated canine parvovirus vaccines are available, they have some limitations. Early in life, maternally derived antibodies protect puppies from canine parvovirus; however, between weaning and successful vaccination there is a period of a few months where puppies are poorly protected (Hernández-Blanco & Catala-López, 2015). Additionally, the immunization schedule is complex, and non-compliance can lead to poor protection in adult dogs (Mylonakis et al, 2016). Similar problems exist in kittens. Jakel et al (2012) have shown that maternally derived antibodies can interfere with vaccinations and that existing vaccines, administered as recommended by the manufacturers, failed more than a third of kittens. The effectiveness of prior arts is limited by these challenges. These problems are circumvented by introducing exogenous peptides that neutralise the virulence and spread of the disease-causing agent into the host via feed without eliciting the host immune response. Moreover, the methods described herein provide scope for the adaptation and refinement of neutralising peptides, which provides synthetic functionality beyond what the host is naturally able to produce.
Antibody heavy chain variable region fragments (VHHs) are small (12-15 kDa) proteins that comprise specific binding regions to antigens. When introduced into an animal, VHHs bind and neutralise the effect of disease-causing agents in situ. Owing to their smaller mass, they are less susceptible than conventional antibodies, such as previously documented IgYs, to cleavage by enzymes found in host organisms, more resilient to temperature and pH changes, more soluble, have low systemic absorption and are easier to recombinantly produce on a large scale, making them more suitable for use in animal therapeutics than conventional antibodies.
Antibodies for Preventing or Reducing Virulence (Summary)
In one aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents to reduce the severity and transmission of disease between and across species. In certain embodiments, the VHH is supplied to host animals. In certain embodiments, the VHH is an ingredient of a product.
Binding to Reduce Virulence
In another aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents, and in doing so, reduce the ability of the disease-causing agent to exert a pathological function or contribute to a disease phenotype. In certain embodiments, binding of the VHH(s) to the disease-causing agent reduces the rate of replication of the disease-causing agent. In certain embodiments, binding of the VHH(s) to the disease-causing agent reduces the ability of the disease-causing agent to bind to its cognate receptor. In certain embodiments, binding of the VHH(s) to the disease-causing agent reduces the ability of the disease-causing agent to interact with another molecule or molecules. In certain embodiments, binding of the VHH(s) to the disease-causing agent reduces the ability of the disease-causing agent to reach the site of infection. In certain embodiments, binding of the VHH(s) to the disease-causing agent reduces the ability of the disease-causing agent to cause cell death.
Antibodies Derived from Llamas
In a further aspect, the present invention provides a method for the inoculation of Camelid or other species with recombinant virulence factors, the retrieval of mRNA encoding VHH domains from lymphocytes of the inoculated organism, the reverse transcription of mRNA encoding VHH domains to produce cDNA, the cloning of cDNA into a suitable vector and the recombinant expression of the VHH from the vector. In certain embodiments, the camelid can be a dromedary, camel, llama, alpaca, vicuna or guacano, without limitation. In certain embodiments, the inoculated species can be, without limitation, any organism that can produce single domain antibodies, including cartilaginous fish, such as a member of the Chondrichthyes class of organisms, which includes for example sharks, rays, skates and sawfish. In certain embodiments, the heavy chain antibody comprises a sequence set forth in Table 1. In certain embodiments, the heavy chain antibody comprises an amino acid sequence with at least 80%, 90%, 95%, 97%, or 99% identity to any sequence disclosed in Table 1. In certain embodiments, the heavy chain antibody possess a CDR1 set forth in Table 2. In certain embodiments, the heavy chain antibody possess a CDR2 set forth in Table 2. In certain embodiments, the heavy chain antibody possess a CDR3 set forth in Table 2.
Antibodies Recombinantly Expressed
In another aspect, the present invention provides a method for producing VHH in a suitable producing organism. Suitable producing organisms include, without limitation, bacteria, yeast and algae. In certain embodiments, the producing bacterium is Escherichia coli. In certain embodiments, the producing bacterium is a member of the Bacillus genus. In certain embodiments, the producing bacterium is a probiotic. In certain embodiments, the yeast is Pichia pastoris. In certain embodiments, the yeast is Saccharomyces cerevisiae. In certain embodiments, the alga is a member of the Chlamydomonas or Phaeodactylum genera.
Antibodies Added to Feed
In yet another aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents and are administered to host animals via any suitable route as part of a feed product. In certain embodiments, the animal is selected from the list of host animals described, with that list being representative but not limiting. In certain embodiments, the route of administration to a recipient animal can be, but is not limited to: introduction to the alimentary canal orally or rectally, provided to the exterior surface (for example, as a spray or submersion), provided to the medium in which the animal dwells (including air based media), provided by injection, provided intravenously, provided via the respiratory system, provided via diffusion, provided via absorption by the endothelium or epithelium, or provided via a secondary organism such as a yeast, bacterium, algae, bacteriophages, plants and insects. In certain embodiments, the host is from the order Carnivora. In certain embodiments, the host is from the order Canidae. In certain embodiments, the host is a domestic dog, wolf, coyote, fox, jackal, or dingo. In certain embodiments, the host is a domestic dog. In certain embodiments, the host is from the family Felidae. In certain embodiments, the host is a domestic cat, a wild cat, leopard, tiger, jaguar, lion, serval, caracal, ocelot, margay, kodkod, oncilla, bobcat, lynx, cheetah, cougar, or jaguarundi. In certain embodiments, the host is a domestic cat. In certain embodiments the host is a non-canine and non-feline species. In certain embodiments the non-canine and non-feline species is a mink, skunk or raccoon.
Feed Product
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHH that bind disease-causing agents and are administered to host animals in the form of a product. The form of the product is not limited. In certain embodiments, the product is feed, pellet, nutritional supplement, premix, therapeutic, medicine, or feed additive, but is not limited to these forms.
Feeding Dosage
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage regime. In practice, the suitable dosage is the dosage at which the product offers any degree of protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal among other factors. In certain embodiments, VHHs are administered to recipient animals at a concentration in excess of 1 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration in excess of 5 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration in excess of 10 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration in excess of 50 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration in excess of 100 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration less than 1 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration less than 500 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration less than 100 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animal at a concentration less than 50 mg/kg of body weight. In certain embodiments, VHHs are administered to recipient animals at a concentration less than 10 mg/kg of body weight.
Feeding Frequency
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents and are administered to host animals as part of a product at any suitable dosage frequency. In practice, the suitable dosage frequency is that at which the product offers any protection against a disease-causing agent, and depends on the delivery method, delivery schedule, the environment of the recipient animal, the size of the recipient animal, the age of the recipient animal and the health condition of the recipient animal, among other factors. In certain embodiments, the dosage frequency can be but is not limited to: constantly, at consistent specified frequencies under an hour, hourly, at specified frequencies throughout a 24-hour cycle, daily, at specified frequencies throughout a week, weekly, at specified frequencies throughout a month, monthly, at specified frequencies throughout a year, annually, and at any other specified frequency greater than 1 year.
Feed Additives
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents and are administered to host animals as part of a product that also comprises other additives or coatings. In practice, the most suitable coating or additive depends on the method of delivery, the recipient animal, the environment of the recipient, the dietary requirements of the recipient animal, the frequency of delivery, the age of the recipient animal, the size of the recipient animal, the health condition of the recipient animal In certain embodiments, these additives and coatings can include but are not limited to the following list and mixtures thereof: meat, a meat by-product, bone meal, fish, fish meal, egg, egg by-product, a vitamin, vegetables, plant matter, plant extracts, an amino acid, a dye, an antibiotic, an antiviral, a hormone, an antimicrobial peptide, a steroid, a prebiotic, a probiotic, a bacteriophage, chitin, chitosan, B-1,3-glucan, vegetable extracts, peptone, krill, algae, B-cyclodextran, alginate, gum, tragacanth, pectin, gelatin, an additive spray, a toxin binder, a short chain fatty acid, a medium chain fatty acid, an omega-3 fatty acid, yeast, a yeast extract, a plant extract, sugar, a digestive enzyme, a digestive compound, an essential mineral, carnitine, glucosamine, an essential salt, fibre, a preservative, a stabilizer, a natural flavour, an artificial flavour, or water.
Non-Feed Uses
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents, and can be used in a non-feed use, such as but not limited to: a diagnostic kit, an enzyme-linked immunoabsorbent assay (ELISA), a western blot assay, an immunofluorescence assay, or a fluorescence resonance energy transfer (FRET) assay, in its current form and/or as a polypeptide conjugated to another molecule. In certain embodiments, the conjugated molecule is can be but is not limited to: a fluorophore, a chemiluminescent substrate, an antimicrobial peptide, a nucleic acid or a lipid.
Antigens
In a further aspect, the present invention provides a polypeptide or pluralities thereof comprising a VHH or VHHs that bind disease-causing agents, produced by a virus of the family Parvoviridae. In certain embodiments, the Parvoviridae virus refers to both current and reclassified viruses. In certain embodiments, the Parvoviridae virus is canine parvovirus type 2. In certain embodiments, the Parvoviridae virus is feline panleukopenia virus.
In certain embodiments, the VHH or plurality thereof is capable of binding to one or more disease-causing agents, originating from the same or different viruses. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to canine parvovirus type 2C capsid protein (SEQ ID NO: 73). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to canine parvovirus type 2A capsid protein (SEQ ID NO: 75). In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to canine parvovirus type 2B capsid protein (SEQ ID NO: 76). In certain embodiments, the disease-causing agent is the canine parvovirus type 2 virus. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to feline panleukopenia virus capsid protein (SEQ ID NO: 74). In certain embodiments, the disease-causing agent is the feline panleukopenia virus. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to mink enteritis virus capsid protein (SEQ ID NO: 89). In certain embodiments, the disease-causing agent is the mink enteritis virus. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to ungulate protoparvovirus 1 capsid protein (SEQ ID NO: 90). In certain embodiments, the disease-causing agent is the ungulate protoparvovirus 1. In certain embodiments, the disease-causing agent is a polypeptide with 80% or greater amino acid sequence identity to mouse protoparvovirus 1 capsid protein (SEQ ID NO: 91). In certain embodiments, the disease-causing agent is the mouse protoparvovirus 1.
The following illustrative examples are representative of the embodiments of the applications, systems and methods described herein and are not meant to be limiting in any way.
While preferred embodiments of the present invention are shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.
Production of Antigens
Recombinant parvovirus capsid can be purified from an insect expression system. For example, capsid protein can be expressed at 26° C. in Spodoptera frugiperda 9 (Sf9) cells cultured in suspension in shaker flasks. Cells are pelleted, frozen, thawed, and then lysed by osmotic shock in a 25 mM sodium bicarbonate solution. The lysate is cleared by centrifugation at 10,000×g for 30 minutes at 4° C. Virus-like particles are precipitated from the supernatant using 20% ammonium sulfate and removed by centrifugation. The pelleted virus-like particles are resuspended in phosphate-buffered saline, pH 7.4 (PBS). Confirmation of virus-like particle production is obtained via electron microscopy and hemagglutination of porcine erythrocytes. The purified virus-like particles are stored at −80° C.
Production of NBXs and Panning
Llama Immunization
A llama is immunized with purified disease-causing agents, these can include recombinantly expressed parvovirus capsid or parvovirus viral particles, which may be accompanied by adjuvants. The llama immunization is performed using 100 μg of antigen at days 0, 21, 42, and 63. At the time of injection, the antigen is thawed, and the volume increased to 1 ml with PBS. The 1 ml antigen-PBS mixture is then mixed with 1 ml of Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA) for a total of 2 ml. A total of 2 ml is immunized per injection. Whole llama blood and sera are then collected from the immunized animal on days 0, 28, 49, 70. Sera from days 28, 49 and 70 are then fractionated to separate VHH from conventional antibodies. ELISA can be used to measure reactivity against target antigens in polyclonal and VHH-enriched fractions. Lymphocytes are collected from sera taken at days 28, 49, and 70.
Panning
RNA isolated from purified llama lymphocytes is used to generate cDNA for cloning into phagemids. The resulting phagemids are used to transform E. coli TG-1 cells to generate a library of expressed VHH genes. The phagemid library size can be ˜2.5×107 total transformants and the estimated number of phagemid containing VHH inserts can be estimated to be >90%. High affinity antibodies are then selected by panning against the antigens used for llama immunization. Two rounds of panning are performed and antigen-binding clones arising from round 2 are identified using phage ELISA. Antigen-binding clones are sequenced, grouped according to their CDR regions, and prioritized for soluble expression in E. coli and antibody purification.
Purification of VHHs from E. coli
TEV protease-cleavable, 6×His-thioredoxin-NBX fusion proteins are expressed in the cytoplasm of E. coli grown in autoinducing media (Formedium) for 24 hours at 30° C. Bacteria are collected by centrifugation, resuspended in buffer A (10 mM HEPES, pH 7.5, 250 mM NaCl, 20 mM Imidazole) and lysed using sonication. Insoluble material is removed by centrifugation and the remaining soluble fraction is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A. The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM Imidazole). The eluted protein is dialyzed overnight in the presence of TEV protease to buffer C (10 mM HEPES, pH 7.5, 500 mM NaCl). The dialyzed protein is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer C. 6×His-tagged TEV and 6×His-tagged thioredoxin are bound to the column and highly purified NBX is collected in the flow through. NBX proteins are dialyzed overnight to PBS and concentrated to −10 mg/ml.
Purification of VHHs from P. pastoris
Pichia pastoris strain GS115 with constructs for the expression and secretion of 6×His-tagged VHH are grown for 5 days at 30° C. with daily induction of 0.5% (vol/vol) methanol. Yeast cells are removed by centrifugation and the NBX-containing supernatant is spiked with 10 mM imidazole. The supernatant is applied to a HisTrap column (GE Biosciences) pre-equilibrated with buffer A (10 mM HEPES, pH 7.5, 500 mM NaCl). The protein is eluted from the column using an FPLC with a linear gradient between buffer A and buffer B (10 mM HEPES, pH 7.5, 500 mM NaCl, 500 mM Imidazole). NBX proteins are dialyzed overnight to PBS and concentrated to −10 mg/ml.
NBX Inhibition of Red Blood Cell Hemagglutination by Parvovirus Capsid
Recombinantly expressed canine parvovirus capsid protein is diluted to approximately 1 nM in PBS and 80 μl, of capsid solution or PBS buffer is added to triplicate wells of a 96-well microtitre plate. 10 μl, of various NBX solutions are added to the capsid solution, yielding final NBX concentrations of 0, 1, 3, 9, 27, 81, 243, 729, or 2187 nM. Capsid/NBX mixtures are incubated at 4° C. for 30 minutes. 10 μl, of a 10% solution of porcine red blood cells (RBC) are added to the wells and mixed. Capsid/NBX/RBC mixtures are incubated at 4° C. for 60 minutes and the plate is photographed. In the absence of capsid protein, the RBCs sink to the bottom of the well. In the presence of capsid protein, the RBCs are agglutinated and remain suspended in the solution of the well. In the presence of neutralizing NBXs, the capsid fails to agglutinate the RBCs and the RBCs sink to the bottom of the well. The minimum hemagglutination inhibition concentration (MHIC) is the lowest concentration of NBX that completely inhibits capsid hemagglutination or RBCs in all three triplicate wells tested.
Table 3 indicates, for all NBXs tested, whether the NBX can neutralize the hemagglutination ability of parvovirus capsid with MHIC values lower than 1 μM or 100 nM
NBX Inhibition of Canine Parvovirus Invasion of MDCK Cells
Madin-Darby Canine Kidney (MDCK) cells are seeded at 4×104 cells per well in a 96-well plate the day before infection. The next day, NBXs are diluted to 50 uM in Eagle's Minimum Essential Medium (EMEM). Canine Parvovirus Type 2 (CPV2) stock is diluted 100-fold in EMEM. NBXs and CPV2 are mixed and the final concentration of NBX is 5 μM. The NBX/CPV2 mixtures are incubated at 37° C. for 60 minutes. Remove media from the cells, add 30 μl of NBX/CPV2 mixtures to the cells, and incubate at 37° C. for 90 minutes. Rock the plate every 15 minutes during the 90-minute infection. Add 70 μl of Dulbecco's Modified Eagle Medium with 2% FBS to the cells and incubate at 37° C. for 24 hours. Fix cells with 4% paraformaldehyde (in PBS) for 60 minutes at room temperature, stop fixation with 0.1 M glycine for 15 minutes. Permeabilize cells with 0.1% Triton-X100 (in PBS) for 30 minutes, and block with 5% milk in PBS-T (0.05% Tween-20) for 60 minutes at room temperature. For detection, incubate with mouse anti-CPV2 antibodies for 60 minutes and HRP-conjugated anti-mouse antibodies for 60 minutes. Incubate with TMB substrate for 30 minutes and stop the reaction with 1 N HCl. Read the absorbance at 450 nm.
Table 4 indicates for all NBXs tested, whether the NBX can neutralize the invasion of MDCK cells by >50% at a concentration of 5 mM.
As used herein, the term “comprise” or variations thereof such as “comprises” or “comprising” are to be read to indicate the inclusion of any recited feature but not the exclusion of any other features. Thus, as used herein, the term “comprising” is inclusive and does not exclude additional, unrecited features. In some embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of.” The phrase “consisting essentially of” is used herein to require the specified feature(s) as well as those which do not materially affect the character or function of the claimed disclosure. As used herein, the term “consisting” is used to indicate the presence of the recited feature alone.
Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well of any dividual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well of any dividual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention, unless the context clearly dictates otherwise.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items.
All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document is specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.
The following references are incorporated by reference in their entirety.
This application claims the benefit of U.S. Provisional Application No. 62/760,765, filed Nov. 13, 2018, which application is incorporated herein by reference. Priority is claimed pursuant to 35 U.S.C. § 119. The above noted patent application is incorporated by reference as if set forth fully herein.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IB2019/001196 | 11/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62760765 | Nov 2018 | US |
